The first molecule possessing antiangiogenic activity was discovered in cartilage by Henry Brem and Judah Folkman in 1975. Since that year more than 300 new molecules capable of inhibiting angiogenesis have been discovered.
In the early eighties, with the discovery of interferon (α/β) as an inhibitor of tumor angiogenesis, clinical experimentation was initiated based on this therapeutic approach.
The media caused quite a stir, when on 3 Mar. 1998 the NewYork Times published the news that two molecules, angiostatin and endostatin, discovered in J. Folkman's laboratories at the Harvard Medical School and Children's Hospital in Boston, were yielding very encouraging results in the struggle against cancer. The high degree of efficacy of these two molecules in inhibiting angiogenesis substantially boosted the search for new compounds. At present, there are about thirty molecules endowed with anticancer activity which act via an antiangiogenic mechanism, which have entered into the clinical trials stage [Phases I-III] and almost the same number of companies and institutions are involved.
In the United States alone it is estimated that there are approximately 9 million patients who could benefit from antiangiogenic therapy. At the present time, at least 4,000 patients have been enrolled into clinical trials using this therapy without any particular unwanted effects being registered.
Within the framework of the general concept of angiogenesis we should distinguish between vasculogenesis, that is to say the formation of blood vessels during embryonic development and angiogenesis in the strict sense of the term, meaning the formation of new blood vessels (capillaries) during the postnatal life starting from pre-existing vessels. The importance of angiogenesis for the growth of solid tumors is amply documented. Over the past three decades it has been reported that tumor growth, as well as the formation of metastases, are strictly dependent on the development of new vessels capable of vascularising the tumor mass.
The inhibition of angiogenesis underlies the formation of necrotic masses within the tumor or the induction of apoptosis in tumor cells.
There are clear-cut differences between neovascularisation in normal tissue and that in tumor tissue. In the former, the vascular endothelium represents a quiescent tissue with a very low mitotic index of its constituent cells (renewal time measured in hundreds of days), and the vascular network is regular, relatively uniform, and suitable for adequately oxygenating all the tissues, without any arteriovenous connection. In tumortissue, on the other hand, stimulation of the proliferation of endothelial cells gives rise to a high mitotic index in the latter (mean renewal time 5 days), the neovascularisation is distinctly irregular with areas of occlusion, sometimes with closed endings, with arteriovenous contacts at some points, and, lastly, the basal membrane presents gaps, which at some points leads to tissue hypoxia. These differences offer the opportunity of identifying drugs which selectively block tumor neovascularisation.
In a tumor, the neovascularisation does not always coincide with a precise stage in the tumor development; there are, in fact, cases in which angiogenysis begins even before the development of the tumor (for example, carcinoma of the uterine cervix), others in which the two phases are coincident (for example, carcinoma of the bladder and breast), and others in which angiogenesis begins after the neoplasm (for example, melanoma and ovarian carcinoma; see, for example, “Manual of Medical Oncology”, IV ed. (1991) G. Bonadonna et al.
Antiangiogenic therapy presents numerous advantages compared to traditional standard chemotherapy (Cancer Research 1998, 58, 31408-16):
a) specificity: its target is a process, i.e. tumor neovascularisation;
b) bioavailability: its target is the endothelial cells, which can be easily reached without the problems of traditional chemotherapy which acts directly on the tumor cells;
c) chemoresistance: this is perhaps the most important advantage of this therapy; in fact, since endothelial cells, unlike tumor cells, are genetically stable, drug resistance phenomena are unlikely to occur;
d) metastatic spread: blockade of the neovascularisation limits the propagation of the tumor cells to other parts of the body via the bloodstream;
e) apoptosis: blockade of the vascular network in the tumor reduces the supply of oxygen and nutrients to the tumor cells; apoptosis is favored in these conditions;
f) reduced systemic tOxicity: toxic effects, such as myelosuppression, gastrointestinal effects and temporary hair loss, which are almost invariably present with traditional chemotherapy, are not observed with antiangiogenic therapy.
A number of molecular elements are known to be involved in tumor angiogenesis (Oncology 1997, 54, 177-84). Pro- and anti-angiogenic endogenous factors are known to be involved in the biological regulatory mechanism in the formation of new vessels.
Among the angiogenic stimulators we should mention: fibroblast growth factors (FGF), vascular endothelial growth factor (VEGF), angiogenin, transforming growth factor-a, tumornecrosis factor (TNF-α), platelet-derived endothelial cell growth factor, transforming growth factor-β, an in-vitro inhibitor, but an in-vivo stimulator, placental growth factor, interleukin-8, hepatocyte growth factor, platelet-derived growth factor, granulocyte colony-stimulating factors, proliferin, the prostaglandins (PGE1, PGE2), GM1-GT1b, substance P, the bradykinins, and nitric oxide.
In contrast, the angiogenesis inhibitors include: the soluble receptor of bFGF, the interferons (α, β, γ), angiostatin, thrombospondin1, prolactin (16 kDa terminal amino fragment), platelet factor 4 (PF4), the tissue metalloproteinase (TIMP) inhibitors, placental proliferinrelated peptide, glioma-derived angiogenesis inhibition factor, the angiostatic steroids, cartilage-derived inhibitor (CDI), the heparinases, 5 interleukin-12, plasminogen activator inhibitor, the retinoids, endostatin, angiopoietin-2, genistein, nitric oxide and GM3.
The integrins are a vast family of transmembrane proteins that mediate cell-to-cell and cell-to-extracellular matrix interactions. All integrins are capable of recognising a common peptide sequence Arg-Gly-Asp (“universal cell recognition site”), though every integrin preferentially recognizes a different conformation of this tripeptide. The inhibition of specific subtypes of integrins can also be of great interest from the pharmacological standpoint for the development of angiogenesis inhibitors.
The control of protein kinase-C (PK-C) may also allow regulation of angiogenesis. There are, in fact, classic PK-C inhibitors capable of completely or partially blocking angiogenesis.
Despite the enormous investments and the involvement of large numbers of institutional and private research centers, the cancer problem world-wide is still far from being definitively solved. Though the prognosis of cancer victims has improved, with survival rates rising over the past 30 years on average from 30 to 50%, and the genetic, cellular and biochemical mechanisms involved in the development of a tumor are now well known, the possibility of defeating or at least limiting this type of pathology is still a problem of keenly felt concern and many aspects are still unsolved, such as the likelihood of recurrence, complete remission and metastatic spread of the primary tumor.
Since the late seventies, when Folkman's observations began to be confirmed by the international scientific community, hundreds of molecules endowed with antiangiogenic activity have been isolated from natural sources (plants, fungi, biological fluids) and synthesized in the laboratory.
A number of drugs are already in Phase III, such as Marimastat (British Biotech.), l'AG3340 (Prinomast-Agouron), and Neovastat (Aeterna), all of which act mainly at the pulmonary level (SNCL) with a mechanism involving interference with the metalloproteinases. Also in Phase III are RhuMad VEGF (this is an anti-VEGF antibody by Genetech) and interferon α (commercial), which are active against solid tumors thanks to their interference with pro-angiogenic growth factors, or TNP-470 (TAP Pharm.) which acts directly on the endothelial cells. Lastly, drugs such as CAl (NCI) and IM862 (Cytran) are active as antiangiogenic agents but with a non-specific and poorly known mechanism.
These above-mentioned drugs may, in a few years, become part of the oncologist's therapeutic armamentarium, but there are other molecules which have recently been inserted in Phase I/II clinical trials such as Combretastatin (OxiGene), methoxy-oestradiol and endostatin (EntreMed), which on the basis of preclinical studies are very promising. Companies such as Bristol Myers-Squibb (with BMS-275291), Novartis (with CGS27023A) or Parke-Davis (with Suramin) are involved in this therapeutic strategy.
Heparin
Heparin is a heterogeneous mixture of naturally occurring polysaccharides of various lengths and various degrees of sulphation which possesses anticoagulant activity and is secreted by the connective tissue mastcells present in the liver (from which it was first isolated), in the muscles, lungs, thymus and spleen.
In addition to the regular sequence, a sequence corresponding to the active site for antithrombin activity has been identified in heparin.
The antitumorand antimetastatic activity of heparin and its derivatives is due to its ability to inhibit heparanase, to block growth factors and to regulate angiogenesis.
Heparan Sulphates (HS)
Heparan sulphates (HS) are ubiquitous protein ligands. The proteins bind to the HS chains for a variety of actions from simple immobilisation or protection against the proteolytic degradation action to specific modulations of biological activities, such as angiogenesis.
The carbohydrate skeleton, in both heparin and the heparin sulphates (HS), consists in an alternation of D-glucosamine (GlcN) and hexuronic acids (Glc.A or IdO.A).
In heparin, the GlcN residues are mainly N-sulphated, whereas in HS they are both N-sulphated and N-acetylated, with a small amount of unsubstituted N—.
HS is also on average less O-sulphated than heparin.
The use of heparin in the treatment of angiogenesis disorders, such as tumors, particularly metastases, is substantially limited by the anticoagulant activity of heparin.
Chemical modifications have been made to heparin so as to reduce its anticoagulant capacity, at the same time preserving its antitumorproperties.
The opening of a unit of glucuronic acid in the antithrombin site reduces the affinity of heparin for antithrombin: in this way, heparins can be used with reduced anticoagulant effects, but still retaining antiangiogenic properties.
Heparanases
“Heparanases” are enzymes belonging to a family of endoglycosidases that hydrolyse the internal glycoside bonds of the chains of heparan sulphates (HS) and heparin.
These endoglycosidases are involved in the proliferation of tumor cells, in metastases and in the neovascularisation of tumors. This suggests they may also be involved in tumor angiogenesis as a result of the release, from the extracellular matrix, of growth factors bound to heparin, such as aFGF (also called FGF-1), bFGF (also called FGF-2) and VEGF.
These enzymes are biological targets for antiangiogenic activity. In the scientific literature there are a large number of structure/activity correlation studies (see, for example, Lapierre F. et al., Glycobiology, vol. 6, (3), 355-366, 1996). Though many aspects have still to be clarified, studies have been reported regarding the inhibition of heparanases by heparin and its derivatives, using specific tests which have led to the emergence of considerations of a structural type which may serve as guides for obtaining new, more selective derivatives.
In the above mentioned work of Lapierre et al., heparin derivatives are described obtained by 2-O desulfation or by “glycol split” (oxidation with periodate and subsequent reduction with sodium borohydride). These derivatives, defined here as “2-O-desulfated heparin” and “RO-heparin”, respectively, have partly maintained the antiangiogenic activity of heparin as assessed by means of the CAM test in the presence of corticosteroids, as reported in Table III (ibid. page 360).
Heparins and FGF
FGFs regulate multiple physiological processes such as cell growth and differentiation, but also functions involved in pathological processes such as tumorangiogenesis.
FGFs are growth factors (a family of more than 10 polypeptides, of which the acid (FGF-1) and basic FGFs (FGF-2) are the ones which have been most studied, which require a polysaccharide cofactor, heparin or HS, to bind to the FGF receptor (FGFR) and activate it.
Though the precise mechanism whereby heparin and HS activate FGFs is unknown, it is known, however, that heparin/FGF/FGFR form a “trimolecular” or “ternary” complex.
One mechanism postulated is that heparin and HS induce so-called sandwich dimerisation of FGF, and the latter, thus dimerised, forms a stable complex with FGFR.
Antimetastatic Activity of Heparin Derivatives
The ability of a primary tumor to generate metastatic cells is perhaps the main problem facing anticancer therapy.
Heparin derivatives with a substantial ability to block heparanase seem to be equally capable of inhibiting angiogenesis both in primary tumors and in metastases.
In addition, the inhibition of heparanase reduces the migration ability of tumor cells from the primary tumor to other organs.
The following table gives an example of structure/antimetastatic activity correlation in the case of heparin:
%inhibitionHeparin97N-succinyl-heparin60N-succinyl-RO-heparin58Low MW heparin86Low MW N-succinyl-heparin61Very low MW heparin83MW = molecular weight
The data in this table suggest that very short fragments of heparin are still endowed with good antimetastatic activity, while this activity is reduced when the amine group of the glucosamine is bound to succinic acid.
The structural requisites of heparin-like molecules that favor the angiogenesis-inhibiting action can be grouped in two categories on the basis of the target one intends to block:
a) blockade of heparanase, an enzyme that hydrolyses the glycoside bonds of the heparan sulphates, releasing growth factors.
To this end the heparin-like compounds preferably comprise sequences of at least eight monosaccharide units containing N-acetyl-glucosamine-glucuronic acid (or N-sulphated glucosamine (see, for example, D. Sandback-Pikas et al. J. Biol. Chem., 273, 18777-18780 (1998) and references cited).
b) blockade of angiogenic growth factors (fibroblast type: FGF-1 and FGF-2; vascular endothelium type: VEGF; vascular permeability type: VPF).
To this end the heparin-like compounds preferably have sequences at least five monosaccharide units long, containing 2-suplhated iduronic acid and glucosamine N, 6-sulphated (see, for example, M. Maccarana et al. J. Bill. Chem., 268, 23989-23905 (1993)).
In the literature small peptides (5-13 amino acids) with antiangiogenic activity (U.S. Pat. No. 5,399,667 of the University of Washington) which act by binding to a thrombospondin receptor, or longer peptides (50 amino acids approx.).
Modified platelet factors are known—(EP 0 589 719, Lilly), capable of inhibiting endothelial proliferation, with IC50=7 nM.
Oligosaccharide fragments with antiangiogenic activity have also been amply described: it has been found, in fact, that by varying the carbohydrate sequence the interaction selectivity can be increased.
In addition, heparin can be used as a vehicle for substances which are themselves antiangiogenic, such as some steroids, exploiting the affinity of heparin for vascular endothelial cells; see, for example, WO 93/18793 of the University of Texas and Imperial Cancer Research Technology, where heparins are claimed with acid-labile linkers, such as adipic acid hydrazine, bound to cortisol. The antiangiogenic effect of the conjugated molecules is greater than that of the unconjugated molecules, even when administered simultaneously.
In Biochim. Biophys. Acta (1996), 1310, 86-96, heparins bound to steroids (e.g. cortisol) are described with a hydrazone group in C-20 that present greater antiangiogenic activity than the two unconconjugated molecules.
EP 0 246 654 by Daiichi Sc. describes sulphated polysaccharides with antiangiogenic activity with Phase II studies.
EP 0 394 971 by Pharmacia & Upjohn—Harvard Coll. Describes hexa-saccharides-heparin fragments—with low sulphation, capable of inhibiting the growth of endothelial cells and angiogenesis stimulated by (FGF-1).
EP 0 618 234 by Alfa Wasserman describes a method for preparing semisynthetic glycosaminoglycans with a heparin or heparin structure bearing a nucleophilic group.
WO 95/05182 by Glycomed describes various sulphated oligosaccharides with anticoagulant, antiangiogenic and anti-inflammatory activity.
U.S. Pat. No. 5,808,021 by Glycomed describes a method for preparing substantially non-depolymerised 2-O, 3-O desulfated heparin with a desulfation percentage in positions 2- of the iduronic acid (1, 2-O) and in position 3 of the glucosamine unit (A, 3-O) ranging from approximately 99 to approximately 75% of the original percentage. This method envisages desulfation conducted in the presence of a cation of a bivalent metal, exemplified by calcium or copper, followed by lyophilisation of the product obtained. The desulfated heparins have antiangiogenic activity.
The aim of the invention described herein is to find optimal glycosaminoglycan structures for generating antiangiogenic activity based on heparanase inhibition and/or FGF growth factor inhibition mechanisms. An additional aim of the invention described herein is to provide a medicament with antiangiogenic activity which is essentially devoid of the typical side effects of heparin derivatives, such as, for example, anticoagulant activity.